Circulating cells can be found in the bloodstream of patients and animal research subjects in many states of health and disease.
For example, circulating tumor cells (CTCs) can be found at some time in all major cancers: ovarian, pancreatic, breast, prostate, colon, renal, and lung cancer. The presence, absence, concentration, or cell counts for CTCs can be used to identify and distinguish patients with cancer, to identify patients undergoing relapse, or to predict survival.
Circulating bacterial cells also are found during early Infection and in stem cell regeneration. In the early stages of infection, there are 1-100 CFU (colony forming units) of bacteria per cc of blood. Bacteria range in size from 0.2-2 microns in width or diameter, and up to 1-10 microns in length for the non-spherical species. Thus, the non-spherical species are comparable in size to circulating tumor cells, while spherical bacteria are smaller. Early detection of infections leads to treatment (antibiotics) prior to systemic collapse. There are 1.2 million cases of blood infection (sepsis) in the US each year, and the cost of treatment runs nearly 9 billion dollars. An early test would allow for early antibiotic selection, reducing cost, and more rapid detection and treatment, improving patient outcome
However, the frequency of these rare circulating cells in the blood is typically on the order of fewer than 100 circulating tumor cells per billion blood cells, and sometimes as rare as 1 in 10 billion. The search for such cells is hard to conceptualize. Imagine trying to locate these cells in 4 billion red cells and 4-10 million white blood cells per cc or human blood. Counting the cells in one cc of blood, by hand, at one cell per second would take in excess of 100 years. This makes finding these rare cells problematic as counting a few hundred particular stars within a galaxy, when real-world issues such as signal to noise are considered.
Approaches have therefore been developed to find these “needle in a haystack” cells. Currently, these approaches in use and under development.
The gold standard known in the art since the 1970s is flow cytometry, which involves flow of cells through an illuminated chamber (Benaron 1982, Cruz 2005, U.S. Pat. No. 4,693,972). Typical flow rates and cell counts in flow cytometry are limited by image and data acquisition times, with cell detection typically down to 1 cell in 1,000 to 10,000 cells (Table 2 in Allan 2010) or, a lower limit of 400,000 to 4,000,000 per cc. This is still 100,000 to 1 million times too insensitive to count an entire milliliter of whole blood, so that for rare cells, an enrichment of the sample of the target cells is usually required-before a flow cytometry assay is performed, in order to get the count times down to, a matter of hours. Often, this enrichment requires the step of attaching targeted magnetic particles to pull out and concentrate the cells. This increase in the concentration of the cells of interest is called an enrichment step, and because this step is both time consuming and lossy, counts are adversely affected as is the time required to perform the test. A key feature of flow cytometry is that the fluid moves, or flows, and that a small volume of the test fluid is monitored at a time. Flow cytometry has even been performed in vivo (US Pub. Pat. Appn. No. 2010/0049041), made possible by flowing cells.
Another known approach is laser scanning (e.g. U.S. Pat. No. 5,547,849). In this approach, cells are placed on a slide or in, stationary capillaries, often with the staining dye present in solution. As the cells are stained in place, there is no washing step. Then, a moving laser beam scans the slide, and a simple confocal detector looking at fluctuations in the amount of light over the small illuminated volume, such as a 10 micron wide capillary tube up to 100 microns deep. Because this was not gathered as an image, noise rejection through image processing could not was not performed. This approach allowed counting of common cells, such as white blood cell T-cell subsets for monitoring of human immunodeficiency virus infections, but the volume of blood monitored made rare cell counting difficult. While this approach reduces prep time, sensitivity is similar to that of flow cytometry, with a lower limit of 400,000 cells per cc, without enrichment or enhancement steps.
A newer and emerging approach is to flow cells through a microfluidics-based microelectromechanical system (MEMS) (e.g., Nagrath 2007). This can allow for cell sorting as well as cell counting. The cells are flowing and are not stationary, and flow rates are limited by the number and diameter of the flow tubes, such that rare cell counting is harder to achieve in large volumes of fluid (e.g., 1 cc). However, such systems have been demonstrated to be able to capture 1 cell in 10,000,000 cells, equal to a lower limit of sensitivity of 400 cells per cc, substantially better than flow cytometry (Table 2 in Allan 2010). To achieve detection of 4-10 cells per cc may still requires an intervening MEMS or laboratory enrichment step.
Other emerging approaches include methods that look for signature proteins (e.g., Proteomics approaches), or lyse the cells and look for signature DNA (e.g., by PCR-based amplification and detection) (Bosolasco 2002). Such approaches are unable to directly enumerate the number of cells, as the target cell has been destroyed, or is not counted; rather, a product or a component of the cell is detected. Further, due to the level of other proteins in the mixture, the detection limit is 1 cell in 10,000 to 1,000,000 cells (or 1 cell in 4,000) (Table 2 in Allan 2010).
All of the above systems lack a method for simultaneous illumination and monitoring of a non-flowing volumetric sample of blood sufficiently large so as to provide counting statistics for rare cells, without any laboratory preparation, separation, or enrichment step.
What is needed is a non-flowing sample device that allows insertion and assay of a volumetric sample of blood sufficiently large to allow for accurate enumeration and/or detection of rare circulating cells, without any laboratory preparation, separation, or enrichment step.